Southern blot hybridizations were used to identify restriction endonuclease fragments from Streptococcus sanguis Challis chromosomal DNA that contain the site of insertion of lactose-specific sequences from cloned fragments of a Streptococcus cremoris lactose metabolic plasmid. The fragments, which include a 15 kilobase pair (kbp) EcoRI fragment, a 6.0 kbp HincII fragment, a 3.2 kbp AccI fragment, a 20 kbp BamHI fragment, a 23 kbp PstI fragment, and two HindIII fragments of 5.0 and 2.5 kbp, are currently being cloned in E. coli to 1) examine their relationship to the lactose-specific sequences, and 2) to replace their regions of homology to the plasmid DNA with a kanamycin resistance gene. The resultant fragments will be excised from the E. coli vector and used to transform lactose S. sanguis strains with selection for kanamycin resistance. Residual homology to the S. sanguis chromosome should permit recombination and replacement of the sequences exhibiting homology to the lactose plasmid. It is expected that hybrid plasmids containing lactose-specific genes will be stably maintained in such a strain, thus permitting further studies on the expression of these lactose genes in a streptococcal host. Chromosomal DNA from a lac+ Streptococcus faecalis strain was also shown to share extensive homology with the cloned lactose PEP-dependent phosphotransferase and phospho-beta-galactosidase genes from the S. cremoris plasmid. More than 150 random and overlapping fragments of a 5 kbp EcoRI fragment from pAMBeta 1, containing the extensively studied broad host-range replicon from this plasmid, have been cloned onto an M13 vector in preparation for DNA sequencing. A S. faecalis protoplast transformation system is also being employed for the cloning of a second, narrow host-range replicon, unable to function in S. sanguis, whose presence in intact pAMbeta1 has been indicated by a series of incompatibility studies using previously obtained natural and subcloned derivatives of pAMBeta 1.